Design and one-pot synthesis of new substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine as potential antitumor agents: in vitro and in vivo studies.

A new efficient and versatile one-pot three-component synthesis of substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives has been developed. It is based on a multistep cascade reaction from 2-aminothiophenes and 2-hydroxy-4-oxobut-2-enoic acids, and derivatives of cyanoacetic acid catalyzed by diisopropylethylamine. As a result, novel pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives (21 compounds) were synthesized in a mild reaction conditions with a high yield. The structures of the developed compounds were confirmed by NMR and elemental analysis. The influence of electron-withdrawing or electron-donor substituents on the antitumor activity of the developed compounds has been identified. In vitro screening analysis of 21 compounds revealed six lead candidates (12aa, 12dc, 12hc, 12ic, 12lb, and 12mb) that demonstrated the most significant antitumor activity against B16-F10, 4T1 and CT26 cells. Necrosis/apoptosis assay showed that apoptosis was the predominant mechanism of cell death. Molecular docking analysis revealed several potential targets for tested compounds, i.e. phosphatidylinositol 5-phosphate 4-kinase (PI5P4K2C), proto-oncogene serine/threonine-protein kinase (Pim-1), nicotinamide phosphoribosyltransferase (NAMPT) and dihydrofolate reductase (DHFR). The lead compound (12aa) can effectively induce cell apoptosis, possesses a high yield (98 %) and requires low-cost starting chemicals for its synthesis. In vivo experiments with melanoma-bearing mice confirmed that 12aa compound resulted in the significant tumor inhibition on 15 d after the therapy. In particular, tumor volume was ∼0.19 cm3 for 50 mg/kg versus ∼2.39 cm3 in case of untreated mice and tumor weight was ∼71.6 mg for 50 mg/kg versus ∼452.4 mg when considered untreated mice. Thus, our results demonstrated the high potential of the 12aa compound in the treatment of melanoma and can be recommended for further preclinical studies.

Encapsulation of a small-molecule drug based on substituted 2-aminothiophenes in calcium carbonate carriers for therapy of melanoma.

Although small molecule drugs are widely used in chemotherapy, their low bioavailability, low-concentrated dose in the tumor zone, systemic toxicity, and chemoresistance can significantly limit the therapeutic outcome. These drawbacks can be overcome by two main strategies: (i) development of novel therapeutic molecules with more significant antitumor activity than currently available drugs and (ii) loading chemotherapeutic agents into drug delivery systems. In this study, we aimed to encapsulate a highly prospective small molecule drug based on substituted 2-aminothiophene (2-AT) into calcium carbonate (CaCO3) microparticles (MPs) for the treatment of melanoma tumors. In particular, we have optimized the encapsulation of 2-AT into MPs (2-AT@MPs), studied drug release efficiency, investigated cellular uptake, and evaluated in vivo biodistribution and tumor inhibition efficiency. In vitro results revealed that 2-AT@MPs were able to penetrate into tumor spheroids, leading to prolonged release of 2-AT. By performing intratumoral injection of 2-AT@MPs we observed significant melanoma suppressions in murine models: ∼0.084 cm3 for 2-AT@MPs at a dose of 0.4 g kg-1versus ∼1.370 cm3 for untreated mice. In addition, the 2-AT@MPs showed negligible in vivo toxicity towards major organs such as heart, lung, liver, kidney, and spleen. Thus, this work provided an efficient strategy for the improved chemotherapy of solid tumors by using an encapsulated form of small molecule drugs.

Synthesis of thieno[3,2-e]pyrrolo[1,2-a]pyrimidine derivatives and their precursors containing 2-aminothiophenes fragments as anticancer agents for therapy of pulmonary metastatic melanoma.

The design and synthesis of new promising compounds based on thienopyrimidine scaffold containing 2-aminothiophene fragments with good safety and favorable drug-like properties are highly relevant for chemotherapy. In this study, a series of 14 variants of thieno[3,2-e]pyrrolo[1,2-a]pyrimidine derivatives (11aa-oa) and their precursors (31 compounds) containing 2-aminothiophenes fragments (9aa-mb, 10aa-oa) were synthesized and screened for their cytotoxicity against B16-F10 melanoma cells. The selectivity of the developed compounds was assessed by determining the cytotoxicity using normal mouse embryonic fibroblasts (MEF NF2 cells). The lead compounds 9cb, 10ic and 11jc with the most significant antitumor activity and minimum cytotoxicity on normal non-cancerous cells were chosen for further in vivo experiments. Additional in vitro experiments with compounds 9cb, 10ic and 11jc showed that apoptosis was the predominant mechanism of death in B16-F10 melanoma cells. With support from in vivo studies, compounds 9cb, 10ic and 11jc demonstrated the biosafety to healthy mice and significant inhibition of the metastatic nodules in pulmonary metastatic melanoma mouse model. Histological analysis detected no abnormal changes in the main organs (the liver, spleen, kidneys, and heart) after the therapy. Thus, the developed compounds 9cb, 10ic and 11jc demonstrate high efficiency in the treatment of pulmonary metastatic melanoma and can be recommended for further preclinical investigation of the melanoma treatment.